This invention relates to low molecular weight components extracted from shark cartilage and to processes for their preparation. This invention also relates to processes for isolating and purifying a cartilage extract under a variety of conditions. The low molecular weight components exhibit at least one of anti-matrix metalloprotease (anti-MMP), antiangiogenic and anti-tumor activities.
Processes for the preparation of shark cartilage extracts and the extracts themselves are disclosed in International Publications WO 95/32722, WO 96/23512 and WO 97/16197. Liquid extracts of shark cartilage have been tested in various assays for antiangiogenic, anticollagenolytic, direct anti-tumor proliferating and anti-inflammatory activities.
WO 95/32722 discloses a process for obtaining a shark cartilage extract having antiangiogenic, in vitro direct anti-tumor proliferating and in vivo anti-tumor activities. That process comprises the steps of blending shark cartilage tissue and reducing the same to a particle size of about 500 xcexcm in water; extracting active components into the water; and fractionating the extracts so obtained in order to recover molecules having molecular weights less than about 500 kDa (0-500 fraction). The liquid cartilage extract was concentrated on a membrane having a nominal porosity of about 1 kDa to form a concentrated liquid extract comprising molecules having molecular weights less than about 500 kDa. The extract was enriched in molecules having molecular weights between about 1-500 kDa. The 0-500 fraction was further fractionated to form a plurality of extracts containing anti-tumor proliferating molecules having molecular weights extending from about 1 to 120 kDa. The WO 95/32722 Publication does not disclose the specific recovery of components having molecular weights less than about 1 kDa. It also does not disclose a process of obtaining a cartilage extract or fractions thereof in organic solvent-containing solutions.
International Publication No. WO 96/23512 discloses a process for extracting biologically active components from any source of cartilage in aqueous solutions. Further, this publication discloses other biological activities associated with the liquid shark cartilage, namely anticollagenolytic and anti-inflammatory activities. The WO 96/23512 Publication does not disclose the recovery of components having molecular weights less than about 1 kDa nor any process making use of organic solvent-containing solutions.
International Publication No. WO 97/16197 discloses a process for the recovery of an aqueous extract enriched in molecules having molecular weights between about 0.1 to 500 kDa. Although that process may recover components having molecular weights of less than about 1 kDa, it does not provide for any recovery of specific low molecular weight components. No component in an isolated or purified form is disclosed.
It is generally accepted in the art that matrix metalloproteases are involved in the processes of neovascularization, promoting the growth of primary tumors and in the formation of metastases. Accordingly, compounds or agents exhibiting antiangiogenic and/or anti-matrix metalloprotease activities are believed to be useful for at least one of inhibiting neovascularization, inhibiting growth in tumors, inhibiting metastatic invasion of cells, inhibiting formation of metastases and treating angiogenesis related diseases.
Given the interest in components obtained from shark cartilage, there exists the need for improved processes for their preparation and for the isolation and purification of other components not previously known to possess biological activity.
The present invention seeks to provide improved processes for the preparation of extracts obtained from shark cartilage.
In one aspect, the present invention provides a process wherein a variety of conditions are used for the preparation of cartilage extracts and fractions thereof containing biologically active components. In one embodiment, the invention provides a process for the preparation of shark cartilage extracts having components possessing at least an anti-MMP activity.
In another aspect, the present invention provides a process by which the 0-500 molecular weight fraction of biologically active components derived from a cartilage liquid extract is separated into two separate fractions wherein the first fraction comprises components having molecular weights less than about 1 kDa (0-1 fraction) and the second fraction comprises components having molecular weights between about 1 to 500 kDa (1-500 fraction).
In order to minimize the formation of component aggregates, to improve the dissolution and the maintenance of a, stable, soluble form, sucrose or one or more other suitable stabilizers such as dextran, Ficoll(trademark), fructose, gelatin, glucose, glycine, inositol, lactose, mannitol and sorbitol can be added in a sufficient stabilizing amount to any of the 0-500, 0-1 and 1-500 fractions, or can be used in any step of the manufacturing process. As used herein in reference to fractions, solutions or extracts, the phrase xe2x80x9ccontaining 1% w/v sucrosexe2x80x9d refers to a respective fraction, solution or extract containing about 1% w/v sucrose. Biologically active components in the 0-1 and 1-500 fractions possess anti-MMP, antiangiogenic and anti-tumor activities.
In another aspect, the present invention provides a shark cartilage derived component having a molecular weight of about 244 amu (atomic mass unit), herein termed -986, possessing at least one of anti-MMP and anti-tumor activities. The process and materials used for the purification of the -986 reveal some physicochemical characteristics of the latter, which are responsible for the partitioning of this component in different solvent phases and chromatographic systems. The present invention also provides a process for the isolation and purification of the -986 component or of an equivalent component obtained from any source of cartilage.
Yet another aspect of the invention provides a purified biologically active compound derived from any source of cartilage which corresponds to the compound having a molecular weight of about 244 amu isolated from shark cartilage and possessing anti-MMP and anti-tumor activities.
Still another aspect of the invention provides a method of inhibiting a MMP enzyme, which method comprises the step of contacting a substrate cleavable by said enzyme with an effective amount of one or more cartilage extracts or fractions derived therefrom or the -986 component prepared according to the present invention.
Still other aspects of the invention provide methods of inhibiting neovascularization and the formation of metastases, which methods comprise the step of contacting a target tissue with an effective amount of a cartilage derived extract, solution, homogenate, suspension, fraction such as the 0-500 fraction, the 0-1 fraction, the 1-500 fraction or the same fractions containing 1% w/v sucrose, or component such as the -986 component.